Genetic stability, genetic variation, and fitness performance of the genetic sexing Salaya1 strain for Bactrocera dorsalis, under long-term mass rearing conditions.

BACKGROUND: A genetic sexing strain (GSS) is an essential component for pest control using the sterile insect technique (SIT). A GSS is developed using a combination of Y-autosome translocation and a selectable marker such as pupal color, resulting in heterozygous males and homozygous females that possess wild-type brown pupae (wp+) and mutant white pupae (wp) alleles, respectively. The genetic sexing Salaya1 strain developed for Bactrocera dorsalis was evaluated using a clean stream and scaled-up for subsequent production lines (e.g., initiation, injection, and release). Colony management under small- and large-scale conditions for long-term rearing may affect the sexing system, genetic background, and fitness performance of the strain. Routine monitoring was applied to study genetic stability, genetic variation, and male mating competitiveness. RESULTS: The percentage of recombinants was significantly different between males (wp) and females (wp+), ranging between 0.21-0.43% and 0.01-0.04%, respectively. Using 106 bands from six ISSR markers, the genetic backgrounds of two generations (F40 and F108) of the clean stream were found to be almost identical (0.960), and between those two generations and the wild population, the similarities were 0.840 and 0.800, respectively. In addition, the sterile males performed well in competitive mating with fertile females (Relative Sterility Index = 0.67 ± 0.13). The rates of fliers calculated from both clean and release streams were higher than 0.95. Regarding the fitness of the Salaya1 strain, the fertility and pupal recovery were similar in all production lines. The sex ratio (Male/Female) distortion was also recorded. CONCLUSIONS: The Salaya1 strain reared at the mass-rearing facility retained its genetic stability, genetic variation, behavior (e.g., competitive mating and flight ability), and traits related to fitness for at least 10 consecutive generations. The filter rearing system is effective at minimising the selection pressure while maintaining the genetic background and fitness performances of the clean stream. These characteristics were stable throughout the production lines. In addition, the production efficiency is comparable among the different production lines and other similar types of GSSs.

Transcribed sex-specific markers on the Y chromosome of the oriental fruit fly, Bactrocera dorsalis.

BACKGROUND: The Oriental fruit fly, Bactrocera dorsalis, is a highly polyphagous invasive species with a high reproductive potential. In many tropical and subtropical parts of the world it ranks as one of the major pests of fruits and vegetables. Due to its economic importance, genetic, cytogenetic, genomic and biotechnological approaches have been applied to understand its biology and to implement the Sterile Insect Technique, currently a part of area-wide control programmes against this fly. Its chromosome complement includes five pairs of autosomes and the sex chromosomes. The X and Y sex chromosomes are heteromorphic and the highly heterochromatic and degenerate Y harbours the male factor BdMoY. The characterization of the Y chromosome in this fly apart from elucidating its role as primary sex determination system, it is also of crucial importance to understand its role in male biology. The repetitive nature of the Y chromosome makes it challenging to sequence and characterise. RESULTS: Using Representational Difference Analysis, fluorescent in situ hybridisation on mitotic chromosomes and in silico genome resources, we show that the B. dorsalis Y chromosome harbours transcribed sequences of gyf, (typo-gyf) a homologue of the Drosophila melanogaster Gigyf gene, and of a non-LTR retrotransposon R1. Similar sequences are also transcribed on the X chromosome. Paralogues of the Gigyf gene are also present on the Y and X chromosomes of the related species B. tryoni. Another identified Y-specific repetitive sequence linked to BdMoY appears to be specific to B. dorsalis. CONCLUSIONS: Our random scan of the Y chromosome provides a broad picture of its general composition and represents a starting point for further applicative and evolutionary studies. The identified repetitive sequences can provide a useful Y-marking system for molecular karyotyping of single embryos. Having a robust diagnostic marker associated with BdMoY will facilitate studies on how BdMoY regulates the male sex determination cascade during the embryonic sex-determination window. The Y chromosome, despite its high degeneracy and heterochromatic nature, harbours transcribed sequences of typo-gyf that may maintain their important function in post-transcriptional mRNA regulation. That transcribed paralogous copies of Gigyf are present also on the X and that this genomic distribution is maintained also in B. tryoni raises questions on the evolution of sex chromosomes in Bactrocera and other tephritids.

The study of the transformer gene from Bactrocera dorsalis and B. correcta with putative core promoter regions.

BACKGROUND: The transformer (tra) is a sex determining switch in different orders of insects, including Diptera, as in the family Tephritidae. The lifelong autoregulatory loop of tra female-specific splicing can be reset by the intervention of male-specific primary signals (M factor). In early development, the functional female and truncated male TRA proteins relay the sexual fates to the alternative splicing of a bisexual switch gene, doublesex (dsx) cascading the sexual differentiation processes. Bactrocera dorsalis (Hendel) and Bactrocera correcta (Bezzi) are among the Bactrocera model worldwide key pests. Area-wide integrated pest management using the male-only Sterile Insect Technique (SIT) relying on genetic sexing systems is effective in control programs. We undertook the molecular characterization and comparative studies of the tra orthologues in the Bactrocera species, including the Salaya1 genetic sexing strain (GSS). RESULTS: RT-PCR revealed that B. dorsalis tra (Bdtra) and B. correcta tra (Bctra) transcripts contained conservation of both constitutive exons and male-specific exons as in other Bactrocera. However, new Bdtra male-specific exons were retained, diversifying the pattern of the male-specifically spliced transcripts. The coding sequences of tra were highly conserved in Bactrocera (86-95%) but less so among related genera (61-65%) within the same Tephritidae family. A conservation of deduced amino acid sequences (18 residues), called the TEP region, was identified to be distinctive among tephritids. The 5' regulatory sequence containing many structural characteristics of the putative core promoter was discovered in B. correcta. The expression patterns of Bdtra and Bctra were sex-specifically spliced and the signals relayed to the dsx genes in the adult wild-types. However, the coexistence of male- and female-specifically spliced transcripts (980 and 626 bp, respectively) of the B. dorsalis wild-type strain was found in the Salaya1 GSS adult males. The Bdtra RNA interference masculinized the XX karyotype females into pseudomales, but their testes were mostly not well developed. CONCLUSIONS: Bdtra and Bctra have sex-specific splicing, similar to Bactroceras, Ceratitis capitata (Wiedemann), and Anastrephas. A newly identified TEP region is proposed in tephritids. A putative core promoter has been discovered in Bctra.

Molecular markers for analyses of intraspecific genetic diversity in the Asian Tiger mosquito, Aedes albopictus.

BACKGROUND: The dramatic worldwide expansion of Aedes albopictus (the Asian tiger mosquito) and its vector competence for numerous arboviruses represent a growing threat to public health security. Molecular markers are crucially needed for tracking the rapid spread of this mosquito and to obtain a deeper knowledge of population structure. This is a fundamental requirement for the development of strict monitoring protocols and for the improvement of sustainable control measures. METHODS: Wild population samples from putative source areas and from newly colonised regions were analysed for variability at the ribosomal DNA internal transcribed spacer 2 (ITS2). Moreover, a new set of 23 microsatellite markers (SSR) was developed. Sixteen of these SSRs were tested in an ancestral (Thailand) and two adventive Italian populations. RESULTS: Seventy-six ITS2 sequences representing 52 unique haplotypes were identified, and AMOVA indicated that most of their variation occurred within individuals (74.36%), while only about 8% was detected among populations. Spatial analyses of molecular variance revealed that haplotype genetic similarity was not related to the geographic proximity of populations and the haplotype phylogeny clearly indicated that highly related sequences were distributed across populations from different geographical regions. The SSR markers displayed a high level of polymorphism both in the ancestral and in adventive populations, and F ST estimates suggested the absence of great differentiation. The ancestral nature of the Thai population was corroborated by its higher level of variability. CONCLUSIONS: The two types of genetic markers here implemented revealed the distribution of genetic diversity within and between populations and provide clues on the dispersion dynamics of this species. It appears that the diffusion of this mosquito does not conform to a progressive expansion from the native Asian source area, but to a relatively recent and chaotic propagule distribution mediated by human activities. Under this scenario, multiple introductions and admixture events probably play an important role in maintaining the genetic diversity and in avoiding bottleneck effects. The polymorphic SSR markers here implemented will provide an important tool for reconstructing the routes of invasion followed by this mosquito.

Gene flow and genetic structure of Bactrocera carambolae (Diptera, Tephritidae) among geographical differences and sister species, B. dorsalis, inferred from microsatellite DNA data.

The Carambola fruit fly, Bactrocera carambolae, is an invasive pest in Southeast Asia. It has been introduced into areas in South America such as Suriname and Brazil. Bactrocera carambolae belongs to the Bactrocera dorsalis species complex, and seems to be separated from Bactrocera dorsalis based on morphological and multilocus phylogenetic studies. Even though the Carambola fruit fly is an important quarantine species and has an impact on international trade, knowledge of the molecular ecology of Bactrocera carambolae, concerning species status and pest management aspects, is lacking. Seven populations sampled from the known geographical areas of Bactrocera carambolae including Southeast Asia (i.e., Indonesia, Malaysia, Thailand) and South America (i.e., Suriname), were genotyped using eight microsatellite DNA markers. Genetic variation, genetic structure, and genetic network among populations illustrated that the Suriname samples were genetically differentiated from Southeast Asian populations. The genetic network revealed that samples from West Sumatra (Pekanbaru, PK) and Java (Jakarta, JK) were presumably the source populations of Bactrocera carambolae in Suriname, which was congruent with human migration records between the two continents. Additionally, three populations of Bactrocera dorsalis were included to better understand the species boundary. The genetic structure between the two species was significantly separated and approximately 11% of total individuals were detected as admixed (0.100 ≤ Q ≤ 0.900). The genetic network showed connections between Bactrocera carambolae and Bactrocera dorsalis groups throughout Depok (DP), JK, and Nakhon Sri Thammarat (NT) populations. These data supported the hypothesis that the reproductive isolation between the two species may be leaky. Although the morphology and monophyly of nuclear and mitochondrial DNA sequences in previous studies showed discrete entities, the hypothesis of semipermeable boundaries may not be rejected. Alleles at microsatellite loci could be introgressed rather than other nuclear and mitochondrial DNA. Bactrocera carambolae may be an incipient rather than a distinct species of Bactrocera dorsalis. Regarding the pest management aspect, the genetic sexing Salaya5 strain (SY5) was included for comparison with wild populations. The SY5 strain was genetically assigned to the Bactrocera carambolae cluster. Likewise, the genetic network showed that the strain shared greatest genetic similarity to JK, suggesting that SY5 did not divert away from its original genetic makeup. Under laboratory conditions, at least 12 generations apart, selection did not strongly affect genetic compatibility between the strain and wild populations. This knowledge further confirms the potential utilization of the Salaya5 strain in regional programs of area-wide integrated pest management using SIT.

Development of a genetic sexing strain in Bactrocera carambolae (Diptera: Tephritidae) by introgression of sex sorting components from B. dorsalis, Salaya1 strain.

BACKGROUND: The carambola fruit fly, Bactrocera carambolae Drew & Hancock is a high profile key pest that is widely distributed in the southwestern ASEAN region. In addition, it has trans-continentally invaded Suriname, where it has been expanding east and southward since 1975. This fruit fly belongs to Bactrocera dorsalis species complex. The development and application of a genetic sexing strain (Salaya1) of B. dorsalis sensu stricto (s.s.) (Hendel) for the sterile insect technique (SIT) has improved the fruit fly control. However, matings between B. dorsalis s.s. and B. carambolae are incompatible, which hinder the application of the Salaya1 strain to control the carambola fruit fly. To solve this problem, we introduced genetic sexing components from the Salaya1 strain into the B. carambolae genome by interspecific hybridization. RESULTS: Morphological characteristics, mating competitiveness, male pheromone profiles, and genetic relationships revealed consistencies that helped to distinguish Salaya1 and B. carambolae strains. A Y-autosome translocation linking the dominant wild-type allele of white pupae gene and a free autosome carrying a recessive white pupae homologue from the Salaya1 strain were introgressed into the gene pool of B. carambolae. A panel of Y-pseudo-linked microsatellite loci of the Salaya1 strain served as markers for the introgression experiments. This resulted in a newly derived genetic sexing strain called Salaya5, with morphological characteristics corresponding to B. carambolae. The rectal gland pheromone profile of Salaya5 males also contained a distinctive component of B. carambolae. Microsatellite DNA analyses confirmed the close genetic relationships between the Salaya5 strain and wild B. carambolae populations. Further experiments showed that the sterile males of Salaya5 can compete with wild males for mating with wild females in field cage conditions. CONCLUSIONS: Introgression of sex sorting components from the Salaya1 strain to a closely related B. carambolae strain generated a new genetic sexing strain, Salaya5. Morphology-based taxonomic characteristics, distinctive pheromone components, microsatellite DNA markers, genetic relationships, and mating competitiveness provided parental baseline data and validation tools for the new strain. The Salaya5 strain shows a close similarity with those features in the wild B. carambolae strain. In addition, mating competitiveness tests suggested that Salaya5 has a potential to be used in B. carambolae SIT programs based on male-only releases.

Evidence of weak genetic structure and recent gene flow between Bactrocera dorsalis s.s. and B. papayae, across Southern Thailand and West Malaysia, supporting a single target pest for SIT applications.

BACKGROUND: Bactrocera dorsalis s.s. (Hendel) and B. papayae Drew & Hancock, are invasive pests belonging to the B. dorsalis complex. Their species status, based on morphology, is sometimes arguable. Consequently, the existence of cryptic species and/or population isolation may decrease the effectiveness of the sterile insect technique (SIT) due to an unknown degree of sexual isolation between released sterile flies and wild counterparts. To evaluate the genetic relationship and current demography in wild populations for guiding the application of area-wide integrated pest management using SIT, seven microsatellite-derived markers from B. dorsalis s.s. and another five from B. papayae were used for surveying intra- and inter-specific variation, population structure, and recent migration among sympatric and allopatric populations of the two morphological forms across Southern Thailand and West Malaysia. RESULTS: Basic genetic variations were not significantly different among forms, populations, and geographical areas (P > 0.05). Nonetheless, two sets of microsatellite markers showed significantly different levels of polymorphisms. Genetic differentiation between intra- and inter-specific differences was significant, but low. Seventeen populations revealed three hypothetical genetic clusters (K = 3) regardless of forms and geographical areas. The genetic structure of sympatric populations slightly changed during the different years of collection. Recent gene flow (m ≥ 0.10) was frequently detected whether samples were sympatric or allopatric. Ninety-five of 379 individuals distributed across the given area were designated as recent migrants or of admixed ancestry. As a consequence of substantial migration, no significant correlation between genetic and geographic distances was detected (R2 = 0.056, P = 0.650). CONCLUSIONS: According to the 12 microsatellite variations, weak population structure and recent gene flow suggest that there is no status for cryptic species between B. dorsalis s.s. and B. papayae forms in Southern Thailand and West Malaysia. Both forms can be treated as a single target pest for the SIT program in an area-wide sense. Additionally, the result of species identification based on molecular data and morphological character are not congruent. The use of independent, multiple approaches in the characterization of the target population may ensure the effectiveness and feasibility of SIT-based control in the target area.

Isolation and characterization of Doublesex homologues in the Bactrocera species: B. dorsalis (Hendel) and B. correcta (Bezzi) and their putative promoter regulatory regions.

Doublesex (dsx) is a double-switch gene at the bottom of the somatic sex-determination hierarchy which regulates sexual dimorphism in many insects. Here, Drosophila melanogaster homologues of dsx were isolated in two Bactrocera species, the oriental fruit fly, B. dorsalis, and the guava fruit fly, B. correcta. Results of RT-PCR analysis suggests that both the B. dorsalis dsx (Bd1dsx) and B. correcta dsx (Bcdsx) genes are transcribed and sex-specifically spliced in accordance with the Drosophila sex-specific splicing mechanism. The cDNA sequences shared a high degree of similarity at the nucleotide level among the Bactrocera species. Structurally conserved domains for DNA-binding and oligomerization were observed in all transcripts suggesting that their proteins function as transcriptional factors for downstream sex-specific gene expression. A purine-rich element (PRE) and four repeat elements (dsxRE) for TRA/TRA-2 binding sites were also found in the 3' untranslated regions (UTR) of both the female Bd1dsx and Bcdsx mRNAs. Notably, a putative core promoter was revealed in Bd1dsx, being probably the first dsx promoter discovered in the tephritid flies.

The utility of microsatellite DNA markers for the evaluation of area-wide integrated pest management using SIT for the fruit fly, Bactrocera dorsalis (Hendel), control programs in Thailand.

The oriental fruit fly, Bactrocera dorsalis (Hendel), is a key pest that causes reduction of the crop yield within the international fruit market. Fruit flies have been suppressed by two Area-Wide Integrated Pest Management programs in Thailand using Sterile Insect Technique (AW-IPM-SIT) since the late 1980s and the early 2000s. The projects' planning and evaluation usually rely on information from pest status, distribution, and fruit infestation. However, the collected data sometimes does not provide enough detail to answer management queries and public concerns, such as the long term sterilization efficacy of the released fruit fly, skepticism about insect migration or gene flow across the buffer zone, and the re-colonisation possibility of the fruit fly population within the core area. Established microsatellite DNA markers were used to generate population genetic data for the analysis of the fruit fly sampling from several control areas, and non-target areas, as well as the mass-rearing facility. The results suggested limited gene flow (m < 0.100) across the buffer zones between the flies in the control areas and flies captured outside. In addition, no genetic admixture was revealed from the mass-reared colony flies from the flies within the control area, which supports the effectiveness of SIT. The control pests were suppressed to low density and showed weak bottleneck footprints although they still acquired a high degree of genetic variation. Potential pest resurgence from fragmented micro-habitats in mixed fruit orchards rather than pest incursion across the buffer zone has been proposed. Therefore, a suitable pest control effort, such as the SIT program, should concentrate on the hidden refuges within the target area.